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Copyright X)1993, AmericanSocietyforMicrobiology

RNA

Duplex

Unwinding Activity

of Poliovirus

RNA-Dependent

RNA

Polymerase

3DP01

MICHAEL W.

CHO,`*

OLIVER C. RICHARDS,' TATIANA M. DMITRIEVA,2 VADIMAGOL,2'3ANDELLIE EHRENFELD'

Department ofMolecularBiologyandBiochemistry, University of California, Irvine, Irvine, Califomnia 92717,1and A. N. BelozerskyInstituteof PhysicalChemicalBiology, MoscowState

University, Moscow119899,2and Instituteof Poliomyelitisand ViralEncephalitides, RussianAcademy ofMedicalSciences, MoscowRegion 142782,3 Russia

Received 28 December 1992/Accepted 13 February 1993

The ability ofhighlypurified preparations of poliovirus RNA-dependentRNApolymerase,3DPo1,tounwind RNAduplex structures wasexamined duringa chainelongation reaction in vitro. Usingan antisense RNA prehybridized toanRNAtemplate,weshow thatpoliovirus polymerase canelongate throughahighly stable RNA duplex of over 1,000 bp. Radiolabeled antisense RNA was displaced from the template during the reaction, and productRNAs whichwereequalinlengthtothetemplatestrandweresynthesized. Unwinding did notoccurinthe absence of chainelongationand didnotrequire hydrolysisof the'y-phosphateof ATP.The rateof elongation through theduplex regionwascomparabletotherateofelongation onthesingle-stranded region of the template. Parallel experiments conducted with avian myeloblastosisvirusreversetranscriptase showed that this enzymewas not able to unwind the RNAduplex, suggesting that strand displacement by poliovirus3DP01 is nota propertysharedbyallpolymerases.

Poliovirus, the prototypic member of the Picomnaviridae

family, has a single-stranded RNA genome of positive

po-larity approximately 7,500nucleotides(nt) long.The genome

is covalently linked to a small viral protein, VPg, at its 5'

terminus(9, 21)andincludesageneticallyencoded

poly(A)

segmentatits 3' terminus(49). Soon after entry of the virus into a host cell, thegenome is released into the cytoplasm

andserves as amRNAtodirect translation ofasingle large polypeptide which is subsequently processed by viral pro-teasesintomatureproteins. Theyinclude structuralproteins

from the P1regionofthegenomeandnonstructuralproteins

from the P2 and P3regionswhich function in theproteolytic

maturation process of viral proteins and in viral genome

replication (for reviews, seereferences 18 and 43).

As a first step in viral replication, the genomic RNA is

copiedtoproduceacomplementaryRNAofnegative polar-ity. Thisreactionis catalyzed by the viralRNA-dependent RNApolymerase,

3DP"",

anenzymebelongingtoa groupof enzymes found only in RNA viruses. Subsequently, this newly synthesized RNA serves as a template to direct

synthesisofdaughter genomic RNAs, therebycompletinga round of replication. Despite long-term efforts in several

laboratories, little is known about the biochemical mecha-nism ofRNA-dependent RNA synthesis (for a review, see

reference36).Invirus-infectedcells, RNA synthesis occurs in replication complexes associated with virus-induced membranevesicles ofrough endoplasmic reticulum origin (2,

5). Replication complexes consist of heterogeneous, multi-stranded structures called replicative intermediates (RI) (4,

11,25)in association with an only partially characterized set of RNA replication proteins. RI contains a core RNA molecule of genome length and various numbers of comple-mentary strands of nascent RNA of various lengths. The core structures of RI, when examined after isolation from

* Correspondingauthor.Electronicmail address:

MCHO@UCI.

EDU

virus-infected cells and deproteinization, appearto contain

significant segments of duplex RNA on the basis of their resistanceto digestionby RNase and electron microscopic

studies (38, 42). However, RNA cross-linking studies sug-gest that, in vivo, RI appears to be predominantly

single

strandedwithnascentstrandsduplexedwith theirtemplates onlynearthe

replication

fork

(38). 3DP°o

is the

only

protein required for elongation of RNA chains in vitro (24, 47); however,currentmodels propose that, in vivo, 3DPoI must function inconcertwith other viraland/or cellularproteins

to accomplishthe complete reaction. These additional pro-teins have beensuggestedtofunction in avarietyof

activi-tiesthoughtto compose partsofthe RNAreplication reac-tion. Forexample, P2-derived proteins (2Corits precursor

2BC) have beenproposed to be involved in the formation

andmaintenanceofreplication complex-associated membra-nous structures as well as in anchoring replication

com-plexes to these structures (3). The involvement of these proteinsin RNAreplication has beenconfirmed by genetic studiesanalyzingseveralmutantsof2B(17) and 2C(22, 27,

32,45).Inaddition,VPg(3B)oritsprecursor(3AB)has been

proposed to function as aprimerfor the initiation of RNA

polymerization (29, 44).

In order for an RNA strand to serve as template for

multiplerounds ofproduct RNA synthesis, the RNA repli-cativemachinery shouldeither preventformationof exten-sivebasepairingbetween thetemplateRNAand the nascent

complementarystrandorcontainahelicaseactivitywhich is able to unwind RNA duplex subsequent to its formation. Suchamechanism would also be necessarytoreleasenewly

synthesized RNAs to serve as templates for subsequent rounds ofreplication, translation, or packaging. Dmitrieva et al.(7)reported that an RNA helicase activity involved in the

synthesis of encephalomyocarditis virus (EMCV)-specific single-stranded RNAwas observed inrelatively crude rep-lication complexesisolated from virus-infected cells. More

recently, they reportedanATP-and RNA elongation-depen-dent RNAhelicase activityofeitherviral orcellularorigin

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which initially copurified with the EMCV RNA polymerase but was lost upon further purification (8). Identification of the viral 2C protein as a potential nucleoside triphosphate (NTP) binding protein by computer-assisted sequence anal-ysis (12-14), in conjunction with the demonstrated implica-tion of 2C in viral RNA replicaimplica-tion, has led to the suggesimplica-tion that thisprotein performs the RNA helicase activity required for the RNA replication reaction. At present, however, no directbiochemical assay of 2C as an RNA helicase has been reported.

Although genetic studies have provided some hints of the roles of some viral proteins in RNA replication, the molec-ular mechanism of the reaction has not yet been established. The enzyme that catalyzes RNA chain elongation,3DP"',has beenpurifiedboth from virus-infected cells (23, 50) and from

bacteria(30, 33)or insect cells(31) expressing the recombi-nantprotein, and itspropertiesarebeginning to be analyzed

(39).As part of ourlong-term studies of the 3D polymerase protein and its functional activities, we examined the RNA duplex unwinding ability of highly purified recombinant

3DP""

during chain elongation in vitro. We report here that this enzyme can unwind a long stretch of RNA duplex so as todisplace strands for continued polymerization of nascent chains and that the unwinding reaction proceeds in an elongation-dependent, ATP-independent manner requiring noadditionalproteins.

MATERIALS AND METHODS

Construction of plasmids. The construction of the plasmids used to generate RNAs to serve as substrates for

3DP"'

unwinding activity assay is diagrammed in Fig. 1. Methods were as described by Sambrook et al. (41). pPOV-3 (16) which contains the3'-terminal segmentofpoliovirus cDNA was digested with BamHI and SalI restriction endonu-cleases.The fragmentcontaining the poliovirus sequence (nt 4600through 3' terminus)wasgelpurified and subsequently ligated into pGEM-4 (Promega) which had been digested with BamHI andSallanddephosphorylatedwith calf

intes-tinalphosphatase. The resulting plasmid, pPV-2C*3D, was usedtogenerateatemplateandaprimerRNA.pPV-2C*3D

wasdigestedwithBglII and SphI, the endswerefilled in with Klenow enzyme, and theplasmidwas religatedtogenerate

pPV-2C*3C*. This plasmid contains poliovirus cDNA se-quence from nt 4600 to 5601 and was used to generate an antisense RNA.

RNA transcription. RNA transcription was performed

with reagents from Promegaaccordingtothe manufacturer's

suggestedconditions with some minor modifications.

Tem-plate RNA (positive polarity) was transcribed from

pPV-2C*3D with SP6 RNApolymerase subsequenttodigestionof the plasmidwithSphI. The primerRNAand the antisense

RNA(negativepolarity)weretranscribed frompPV-2C*3D

and pPV-2C*3C*, respectively, with T7 RNA polymerase subsequent to

digestion

of the

plasmids

with ScaI and

EcoRI, respectively. To generate 32P-labeled antisense RNA,

[ot-32PjUTP

(Amersham)

was added tothe

transcrip-tion reactranscrip-tionto afinal

specific activity

of 12mCi of UTP per mmol. Inaddition,theconcentration of unlabeled UTPwas

lowered by 10-fold to0.1 mM,while the concentrations of the other nucleotides were kept at 1 mM.

Subsequent

to transcription at 37°C for 2h, the DNAwas

digested

with

RQ1 DNase

(Promega)

at 37°C for 30 min. RNA was

extracted with

phenol-chloroform

andwas

precipitated

with ethanol with 2 M ammonium acetate as a salt to minimize

coprecipitation of

unincorporated

nucleotides. The

pellet

H I4600

glll5601

EcoR I

OR' BA9LIan5HI

tI pPOV-3 rpGEM4 JSalIJr ~~~~~~~~~~~Sph'I

SalIDigest BamHIDigest

I

Sca1 7332 CIP

Sal I Digest BamH IDigest

oigation

N\EcoR I (Apr '~(~ BamHI

{AM

pPV 2C*3D

OR Bg~slII

SphlI

ScaI Bgl Digestof

SphIDigest

Klenow

MEVectoraReligation

\ ~~~~~EcoRI

AApr N BamHI

[image:2.612.322.558.80.436.2]

pPV2C3CX

FIG. 1. ConstructionoftheplasmidsusedforsynthesizingRNA transcripts. A BamHI-SalI fragment of pPOV-3 containing the poliovirus sequence was ligated into the multiple cloning site of pGEM-4 to generate pPV2C*3D. pPV2C*3D was subsequently digestedwithBgllland

SphR,

the ends werefilledin,andthevector was religated to generate pPV2C*3C*. The nucleotide numbers indicatedonpPOV-3refertothenucleotideresidues of the

poliovi-rus

genome.

was

resuspended

in TEbuffer

(10

mMTris-HCn

[pH 8.0],

1 mM

EDTA)

andstored at -20wC.

RNAsubstrate

preparation.

RNA substrates wereformed

by

incubating

mixtures of RNAs at65hC for 30 min in TE buffer

containing

100 mMNaCl.

Approximately equal

molar amountsof all three RNAswere derived

by

experimentally

titrating

the

hybridization

reactionsothat the

mobility

of all detectable

single-stranded

template

RNAwasshiftedtothat of

duplex

RNA on an agarose

gel.

No excess

primer

or antisense RNAswereapparentafter

staining

with ethidium bromide. RNA substrates were

subsequently

stored at -20RC. To

prepare

RNAsubstrateswithout detectable lev-els of nucleotide

contamination,

these RNA

preparations

were

subjected

to a second ethanol

precipitation

with 2 M ammoniumacetate.

To examine the

degree

of base

pairing

between the

tem-plate

strand and either the antisense strand orthe

primer,

RNAwastreated with RNaseA

(10

,ug/ml)

at30°Cfor 30 min in TE buffer

containing

300 mM

NaCl,

and the

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resistant duplexes were analyzed by electrophoresis in an agarose gel.

Purification of poliovirus polymerase 3D. PoliovirusRNA polymerase wasexpressed in Escherichiacoliharboring the plasmid, pEXC-3D, at 30°C, and crude sonicates were prepared as described by Rothstein et al. (40). The lysates were clarified by centrifugation at 100,000 x g, and poly-merase waspurified from the supernatant (S-100),basically as described previously (31). The purification protocol in-cluded 0 to40% saturation ammonium sulfateprecipitation, phosphocellulose (Whatman), Mono-Q (Pharmacia LKB Biotechnology), andPhenyl-Superose (Pharmacia LKB) col-umnchromatography. As afinal step, the enzyme was bound and eluted from apoly(U)-Sepharose 4B (Pharmacia LKB) column (1.5 by 8 cm) in 50 mM Tris-HCl (pH 8.0)-5 mM

3-mercaptoethanol-0.1%

NonidetP-40-10%

glycerol,

witha 100-ml gradient from 0.05 M KClto0.4 MKCl.

Forall purification steps the presence ofpoliovirus poly-merase was monitored by assaying poly(U) polymerase

activity on a poly(A) template as described by Hey et al. (16). For the final purification step, polymerase puritywas evaluated by analysis of column fractions in a sodium dodecyl sulfate(SDS)-10% polyacrylamide gel and detection ofprotein by silver staining(28).

Poliovirus polymerase and AMV RT assays, and product

analysis.

Approximately0.25 ,ug oftemplate RNA and 30 ng ofpurifiedpoliovirus polymerasewereincubatedat30°C for 1 h under the following conditions unless notedotherwise: 50 mM HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid; pH 8.0), 0.5 mM each NTP (Boehringer

Mannheim), 4 mM dithiothreitol,3 mM magnesium acetate, 0.06mMZnCl2,4,ug ofactinomycinDperml, and 0.8 U of RNasin (Promega) per ,ul. In some experiments, 5'-adeny-lylimidodiphosphate (AMP-PNP; Boehringer Mannheim) was used at 0.5 mM. In experiments in which de novo-synthesized RNAwas labeled,

[a-32P]UTP

was added to a finalspecificactivity of4mCi/mmol,andapproximately 1 ,ug of RNAtemplatewasused.Polymerasereactions withavian

myeloblastosis virus (AMV) reverse transcriptase (RT; Promega)wereperformed according to the manufacturer's

suggestedconditions with either 1or0.75 mM deoxyribonu-cleotidesateither 30or42°C. ProductDNAwaslabeled with

[a-32P]dATP

(1.3 mCi/mmol; Amersham). Reaction products wereanalyzed on1% agarose gels under either nondenatur-ing or glyoxal-denaturing conditions as described by Sam-brook etal. (41).

RESULTS

RNA substrates for RNA duplex unwinding assays. To examine RNA duplex unwinding activity of poliovirus

RNA-dependent RNA polymerase, we used the strategy previ-ously developed by Dmitrieva et al. (8), applied to a different setofRNAsubstrates. The assay involves a polymerization reaction with an RNA template prehybridized to a segment of complementary, antisense RNA. The antisense RNA serves asanobstacle for apolymerase. RNA duplex

unwind-ingactivity allows displacement of the antisense RNA from thetemplate RNA. The length of the resulting polymerized

product is equal to that of the template RNA if unwinding occurs; a shorterproduct, equal to the length of the unhy-bridized segment of the template, results if the antisense RNAis notunwound and displaced.

Figure 2 showsadiagram of the template RNA (positive

polarity), a template-specific primer RNA (negative

polari-ty),and the antisense RNA (negative polarity), synthesized

i IliC D

I^

IS ..

CI

T ....

Template .102lt |

-Primer 278nlt

I

[image:3.612.333.535.81.158.2]

.I

FIG. 2. Diagramof transcripts usedtogenerate RNAsubstrates. Thetemplateand primerRNAsweretranscribed frompPV2C*3D subsequenttodigestion oftheplasmidwithSphI andScaI, respec-tively. The antisenseRNA wastranscribed frompPV2C*3C* sub-sequent todigestion oftheplasmidwithEcoRI.Thelengthsand the polarity of the transcripts are as indicated. El, E, and _ denotepoliovirussequences, poly(A)or poly(U)tract, andvector

sequences,respectively.Thecorresponding regionsof the

poliovi-rus genome areillustratedatthe top.

toproduce substrates for the RNAduplex unwindingassay. The corresponding regions of the poliovirus genome are indicated at the top of the figure. The template RNA is

approximately3,022ntlong, including 2,841ntofpoliovirus

RNAsequences,approximately50ntofpoly(A) tail, plus30 and 101 nt ofvectorsequences at the5' and 3' ends of the RNA,respectively.TheprimerRNAis 278ntlong,

contain-ing 106ntofpoliovirusRNA sequences,approximately50nt

of poly(U) tail, and 122 nt of vector sequences. The

an-tisense RNAis1,047ntlong,including 1,006ntofpoliovirus

RNAsequences,and 21and 20ntofvectorsequencesatthe 5' and 3' ends of theRNA, respectively. Becausethereare 21 ntofvectorsequence atthe 5' terminus of the antisense RNAwhich do not hybridizewith the template, theduplex

formed between thetemplateand the antisense RNA isonly

1,026bp.

Thetemplate-specificprimerRNAwasdesignedtoanneal tothe 3'end of thetemplateandtoextendslightlybeyond it,

since it contained ashort segment(21 nt)of

noncomplemen-tary RNA at its 5' terminus. This prevented the template

from forming a hairpin turn at its own 3' terminus which would allowself-primed RNAsynthesis to occur. We have observed that any template containing a heterogeneous

sequence at its 3' terminus supports primer-independent synthesis thatproduces asnapback product approximately

twice the length of the template (see below). RNAswhich arepolyadenylated attheir 3' termini donotself-prime; the presence of even as few as six heterogeneous nucleotides

following the poly(A) tail, however, results in self-primed

RNAsynthesis bythepoliovirusRNApolymerase (datanot

shown).

Thetranscripts shown in Fig. 2 were annealed in various combinations to produce substrates for the RNA duplex

unwinding assay. A schematic diagram and an ethidium

bromide-stainedgelof these substrates are shown in Fig. 3. Substrate1(lane1)is theRNAtemplatealone.Incubation of thetemplatetogether with primer (substrate 2) resulted in a detectable shift in itsmobility (lane 2), indicating the forma-tion of a partial duplex. The larger duplex region formed between the template and antisense RNA (substrate 3) causedanevengreatermobility shift(lane 3). Substrate 4 is

composedoftemplate,primer, and antisense, and its

mobil-itywasretardedevenmore(lane 4). Substrates 5 and 6 (lanes 5 and 6) are the same as3 and 4 except that the antisense RNA waslabeled with

32p

(indicated byathicker line in the

diagram in Fig. 3). These were prepared to allow direct measurement of the displacement of the antisense RNA

Antistrise 047 iit 1-_-.---I

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M 3DPOl

I

3 . _M (Kd)

(Kbp)

3.1

-2.0

-

1.6-I.(.)

0.5

-NI 4 6 NI

66-

a-45- _

[image:4.612.83.277.80.238.2]

36- ^_

FIG. 3. Substrates for RNA duplex unwinding assay. Six RNA substrateswere generated, asdiagrammed schematically at the top, by annealingvarious combinations of transcripts shown in Fig. 2. Thesesubstrates were analyzed on a nondenaturing agarose gel and visualized after staining the gel with ethidium bromide. Double-stranded DNA markers areshown in lanes M. The antisense RNA usedtogeneratesubstrates 5 and 6 was labeled with[32P]UTPand isindicated by a thicker line in the diagram.

during the unwinding reaction. Treatment ofsubstrate 6 with RNase A under conditions of high salt specific for the digestion of single-stranded RNAs generated a sharp, RNase-resistant, radiolabeled band of approximately 1,026 bp, indicating stable base pairing between the templateand the antisense RNAs (data not shown). In addition, an ethidiumbromide-stainedband of approximately 300 bpwas observed, representing the template-primer duplex region (datanotshown).

Poliovirus RNApolymerase.We have previously described thepurification andcharacterization of the poliovirus RNA-dependentRNApolymerasefrom clones ofE. coli harboring theplasmid pEXC-3D (31). This plasmidencodes the polio-virus 3CD protein driven bythetrppromoter(37). Induction ofexpressionof3CD results inautocatalyticcleavage by the 3C protease sequence to generate the 3D polymerase pro-tein, identical to thatproduced after infection of cells with poliovirus. SDS-polyacrylamide gel electrophoresis (PAGE) analysisof thepurified proteinused in thesestudies is shown inFig. 4. Nocontaminatingproteinsweredetectedbysilver

staining.

Polioviruspolymerasecanelongate throughalargesegment of RNA duplex. To determine whether an antisense RNA

hybridizedtothetemplatepresentedastable obstacletothe poliovirus polymerase,the RNAsubstrates described inFig. 3 were utilized in a standard polymerase elongation assay containing [32P]UTP, and theproductswereexaminedon a

denaturingagarosegel. Figure5 showsan autoradiogramof the reaction products. When the template alone (substrate 1),withnoprimerandnoantisense RNA,waspresentedas a substrate to the purified poliovirus RNA polymerase, a

predominant product of approximately 6,000 nt was made

(lane 1). Thisproduct is approximatelytwice the lengthof the template and indicated that this template could

self-prime, presumably bycreatinga hairpin structure at the 3' terminus that allowed the synthesis ofa snapback RNA in which thenewly synthesized polynucleotidewascovalently attached to the template. Annealing the antisense RNA to thistemplate (substrate 3)hadno effect onthe self-priming capacity of the template and, more importantly, had no

29-24- a..m

20- N

DF

--FIG. 4. SDS-PAGE analysis of purified poliovirus polymerase. Poliovirus polymerase was purified according to the protocol de-scribed in Materials and Methods. Approximately 300 ng of the purifiedenzyme wassubjectedtoSDS-PAGE(10%acrylamide) and subsequently analyzed by staining the gelwith silver. Molecular weightstandards are shown in lane M, and the dye front is indicated asDF.

effectonthe length of the product(Fig. 5, lane 2), suggesting that the antisense RNA was unwound anddisplaced from the

template allowing polymerization of new product RNA

complementaryto the5' end of the template. Addition ofa primer to these substrates (to generate substrates 2 and 4) produced theproducts shown in Fig. 5, lanes 3 and 4. Some

self-primingstilloccurred,mostlikelyfromexcesstemplates nothybridizedtoprimer.This small amount ofunhybridized templatewould not have been detectableby ethidium bro-mide staining in Fig. 3. The major product, however, was about3,000ntinlength, equaltothelength of the template.

Again,nodifference inproduct lengthwasobserved whether or not antisense RNAwasannealed tothetemplate. These data demonstrate that the poliovirus polymerase elongated

through the region of 1,000 bp presentedby the template-antisense duplexregionand suggest that the enzyme could unwind the duplex. Two small bands migrating near and below1,000nt wereobserved in lanes 2 and 4(Fig. 5).These products appeared when antisense RNA in the absence of

templatewaspresentedassubstrates (datanot

shown),

but theexact nature of these bands hasnotbeen investigated.

Parallelexperimentswereconducted with AMV RT

(Fig.

6),

which has been

previously

showntobe unabletodisplace complementary RNAfroman RNA

template

(6).Thesame

template-primer substrates, in the presence and absence of antisenseRNA(substrates2and4)wereusedtodirect DNA

synthesiswith AMVRT at30°C.Asa

control,

lane1

(Fig. 6)

shows thepredominantly 3,000-nt product of RNA

synthe-sizedbythepolioviruspolymerasewith substrate

2,

asseen

before in Fig. 5. Small amounts of the

self-primed,

dimer-length product are also visible. RT

produced

a

full-length

cDNAof thesame

3,000-nt

size

(Fig. 6,

lane

2);

in

addition,

anotherproductjustover

2,000

ntin

length

is apparent,most

--o-

3DPOI

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[image:4.612.370.509.85.315.2]
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RNASubstrate

e..

1 3 2 4

A?..o

#0_

.... *.f

2 2 4 4

---3000nt ---2000nt

-.i.

.71.W.M.W

io

400 iw

.&- --*-lOOOI nt

A 1 2 3 4 A

FIG. 5. RNA synthesis by poliovirus polymerase. RNA sub-strates1, 3, 2,and 4(schematicallyillustrated in thebox)wereused in a3D1') polymerization assayinthe presenceof[32P]UTP. RNA products were denatured with glyoxal and subjected to electro-phoresison anagarosegel,inlanes1to4. Anautoradiogramof the driedgelis shown. The antisense RNA(lanesA; -1,000nt)and the template RNA (not shown; -3,000 nt) were used as mobility markers. The approximate length of the 6,000-nt product was

estimatedfrom itsmobility relativetothemarkers.

likelythe result ofapause (strongstop)by the enzyme due to secondarystructurepresentin thetemplateRNA. When the AMV RT was presented with substrate 4, which con-tained hybridized antisense RNA, the enzyme was

appar-entlyunabletounwind theduplexanddisplacethe antisense

strand, andsynthesis terminated atthe block. This resulted inapredominant cDNA product ofapproximately2,000ntin

length, asseeninFig. 6, lane 3. A shorter product of about 1,000ntwasalsodetected, presumably dueto a RTpause or theantisense RNAasdescribed above(Fig.5, lanes 2 and4).

Identical results were obtained when reactions were per-formedat42°C(thenormal reactioncondition for AMV RT; data not shown), suggesting that the reduced temperature was notthe reason for the inabilityofAMV RTtoelongate

through the RNAduplexregion.

Addition of poliovirus polymerase to the RT reaction did not affect the length of the cDNA product (Fig. 6, lane 4). This result allowed us to exclude the possibility that the RNA unwinding observed in the poliovirus polymerase-catalyzed reaction was due to a contaminating helicase

activityinthe purified enzyme preparation. If a contaminant was unwinding the duplex so as to enable the poliovirus enzyme to elongate to the end of the template, then its unwinding activity would also have allowed RT to synthe-size a full-length product. Thus, the unwinding activity observed in the poliovirus polymerase reaction was inherent inthe enzyme itself.

DisplacementofantisenseRNA from the template. In order todemonstrate directlywhetherpoliovirus polymerase can unwind the antisense RNA from the template, we performed

A 1 2 3 4 A

FIG. 6. DNAsynthesis byAMV RT. RNAsubstrates2 and4 (shown in thebox)wereusedtodirect DNAsynthesis with AMV RT (lanes 2 and 3, respectively). The newly synthesized DNA products werelabeled with[32P]dATP. The reaction productswere denatured with glyoxal and subjected to electrophoresis on an agarose gel. An autoradiogram of the dried gel is shown. Lane 1, RNA products synthesized by poliovirus polymerase with RNA substrate2as acontrol;lane4,polioviruspolymerasewasaddedto the RT reaction shown in lane 3. Approximate lengths of the antisenseRNA(lane A;-1,000nt)andmajor products (-2000 and -3000nt)areindicated.

an antisensedisplacement assay. Inthis assay,weused the substrate (Fig. 3, substrate 6) containing primer and radio-labeled antisense RNA, in a polymerization assay with unlabeled nucleotides. The rationale for this assay is that the antisense RNA, when unwound from the template by an advancing polymerase, would be displaced from the tem-plate, and theradioactivityin thesubstrate woulddisappear

with time. Concomitantly, a corresponding signal would appearasfreeantisense RNA. Sampleswereremoved from apolymerization reactionatvarious timesduring the course of the assay, and theproductswereanalyzedon a

nondena-turingagarosegel.Figure7Ashowsanautoradiogramof the gel. Reaction products from 0, 15, 30, 60, 90, and 120 min of incubation are shown in lanes 1 to 6, respectively. As

expected, the radioactivity in the substrate gradually

de-creased, accompanied byan accumulation ofaband

comi-grating with the antisense RNA. The kinetics of antisense

displacement correlated with thekinetics of RNA

polymer-ization in the same reaction. The displaced product in-creasedlinearlywithtimeduring the first 60 min but reached a plateau thereafter. This shows directly that the antisense strandwasunwound andreleased from the template. Omis-sion of the poliovirus polymerase from the reaction incu-bated for 120 min resulted inastable RNAduplex,withno

displacement of the antisense RNA (lane 7). Inaddition to free antisense RNA, a minor product that migrated more

slowlythan the substrate appearedduringthecourseof the

reaction, butnotin reactions in which the polymerase was

1-2

4=-3DPOI

RT

RNA

r2 -L.I

i'4-I"

---600()nt

4".am --*--3000nt

-.*-

-IOOOnt

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[image:5.612.101.253.81.320.2] [image:5.612.351.516.81.319.2]
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RNA DUPLEX UNWINDING BY 3DP0' 3015

I,

.i

+3DP0'6

-0' 15' 30' 60' 90'120'120' -+ ++ +- 3DPOIRT .5\ 2

-

).-anOnam m

_o

_

A 1_2 4 5 6 7A A 1 2 3

FIG. 7. Antisense RNA displacement assay. RNA substrate 6 (Fig. 3) containing a radiolabeled antisense RNA was used in polymerizationassays, with eitherpoliovirus polymerase or AMV RT. (A) Kinetic analysis of RNA duplex unwinding activity by poliovirus polymerase. Reaction products from the indicated times of incubation with(lanes 1 to 6) or without3DP"" (lane 7, 120min) wereanalyzed on a nondenaturing agarose gel. An autoradiogram of thedried gel isshown. The antisense RNA (lanes A) is used as a mobility marker. The positions of the RNA substrate, reaction intermediate, and displaced antisense RNA are indicated on the rightside of the panel. (B) Products of RT reaction (1-h incubation at30°C) in whicheither RT alone (lane 1), RT and poliovirus3DP°o (lane 2),orpoliovirus 3DP°' alone (lane 3) was used. The positions of theRNAsubstrate and reaction product are indicated on the right sideof the panel.

omitted. This product comigrated with the fully duplexed final product of the polymerization reaction, and thus it appears tobe an intermediate in which the polymerase has synthesized up to the 5' terminus of the antisense RNA. During the polymerization reactions, not all of the templates were transcribed. In general, approximately 50% of the templates were converted to product during 120 min of reaction. Somewhat less than50% of the antisense RNA was recovered as a discrete band on the gel; some of the

antisense RNAoccurs asduplex intermediate asdescribed

above, and it ispossiblethatsomemightbedegraded.

Theantisense RNA migrates astwobands in a

nondena-turingagarosegel(Fig.7A, laneA).Thedistribution of RNA in each band was concentration dependent with higher

concentration favoring the more slowly migrating band. Heating the sample immediately prior to electrophoresis reduced the proportion which migrated more slowly. The upperband thus appearstobealooselybase-paired dimer;it

migrates just above a 1,018-bp DNA marker and it was sensitivetodigestionwith RNase A under saltconditions in which stable duplexes were resistant and single-stranded

RNAswere digested(datanotshown).

Antisense displacementwas also examined after reverse

transcriptionofthesamesubstrate with AMV RT

(Fig. 7B).

Lane 1 shows that although the amount of label from the substrate band decreased, no antisense RNAwas released

duringthereversetranscription. Instead, aband of

approx-imately 1,000 bpwasgeneratedpresumably bythe RNase H

activity of RT which hydrolyzed the RNA strand of the RNA-DNA duplex formed after cDNA

synthesis, thereby

releasing the remaining template-antisense RNA duplex.

The same product profile was observed when

poliovirus

polymerase was added to the RT (lane

2),

also

suggesting

A 1 2 3 4 5 6 7 8 9 A

FIG. 8. NTP requirements of unwinding activity by poliovirus polymerase.RNAsubstrate 6 (Fig. 3) was used in an RNA unwind-ingassaywith either no NTP(lane 1), all four NTPs (lane 2), or three NTPsexcludingATP,CTP,GTP, or UTP (lanes 3 to 6, respective-ly).Products were analyzed on anondenaturing agarosegel; next cametheautoradiographicexposureof thedried gel. Lane 7 shows

areaction in which AMP-PNP alone was present. CTvP,GTP, and UTP(lane 8)or all four NTPs (lane 9)were addedin addition to AMP-PNP. TheantisenseRNA(lanes A) is usedas amarker. The bracketshowsasmearof reaction intermediates which accumulated inlane3.

that the poliovirus polymerase preparation does not have a

contaminatingRNAhelicase. Lane 3 shows poliovirus poly-merase alone under DNA synthesis reaction conditions for RT asanegative control.Asimilar pattern was attained from reaction in which neither RT nor poliovirus polymerase was present(data notshown).

Unwinding depends on chain elongation but not ATP

hy-drolysis.

To determine whether duplex unwinding required RNA polymerization, strand displacement was measured with substrate 6(Fig. 3) under conditions which precluded RNAsynthesis. When all ribonucleotides were omitted from the reaction, no unwinding occurred (Fig. 8, lane 1).

Sup-plementationwithall four ribonucleotides restored

unwind-ingand antisensedisplacement(lane 2),butadditionof only three did not (lanes 3 to 6). The absence of unwinding activityinthese reactionsadditionallysupportstheprevious

conclusion that contaminating helicase activity was not present in our purified enzyme preparation. Some activity

wasobserved in reactions which lacked onlyATP(lane 3);

thiswas likely due to minor contaminations of ATP in the mixture ofCTP,GTP, and UTP.Reducingthe NTP concen-trationin the reaction from 500to25 ,uM eliminated the low level ofactivity observed in the absence of ATP (data not

shown). Ribonucleotide titrations showed that RNA

poly-merization and duplexunwinding occurred, albeit less

effi-ciently,with NTP concentrationsaslow as5 ,uM,which is

100-foldlowerthan thestandardreaction conditions.

Thus,

only low levels of ATP contamination in the other NTP

preparationsat500,uMcouldsupplysufficientATPforsome activity to be observed. Under the conditions of

limiting

reaction, asmear ofintermediate RNA

products

were seen to accumulate above thetemplateband

(lane 3, brackets).

The experiments described above demonstrated that

hy-drolysis of ATP or other NTPswas notsufficient for

poly-merase-catalyzed RNA

unwinding.

To determine whether

hydrolysis of the y-phosphate of ATP was

required

in

addition to chain

elongation,

a

nonhydrolyzable

analog

of

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ATP,

AMP-PNPwassubstituted for ATP in the reaction. No

unwinding

occurred in the presence of AMP-PNP alone

(Fig.

8,

lane

7); however,

full

activity

was restored when

CTP,

GTP,

and UTP

(lane 8)

orwhen all four NTPs

(lane 9)

were

added to the AMP-PNP reaction

(compare

to lane

2).

Un-winding

assaysdoneat25,uMNTPtoexclude the

possibility

that the

unwinding activity

wasdueto minor contaminants of ATP in

CTP, GTP, UTP,

and AMP-PNP

preparations

also revealed that AMP-PNP could substitute for ATP

during

unwinding activity (data

not

shown).

These

experiments

show thatwhile RNAchain

elongation

is

required

for RNA

duplex unwinding activity, hydrolysis

of ATP is not

re-quired.

DISCUSSION

Many

viruses that infect

bacterial, animal,

or

plant

cells contain their

genetic

information in a

single-stranded

RNA genomeof

positive

polarity.

These viruses encodean

RNA-dependent

RNA

polymerase

which

catalyzes

the

unique

reaction of RNA

synthesis

from a

single-stranded

RNA

template. Replication

of viral RNA takes

place

intwostages:

synthesis

ofa

complementary

(negative polarity)

RNAwith the viral genomeas

template,

and then

synthesis

of progeny

virus

genomic

RNAwith the

negative-strand

RNA as

tem-plate.

In the case of

poliovirus

RNA

replication,

RNA

synthesis

occurs via

partially

double-stranded structures

called

replicative

intermediates. To prevent accumulation of dead end

products

in which

newly synthesized

RNAstrands are

extensively hybridized

to their

templates,

the nascent

strand must be

prevented

from base

pairing

with the

tem-plate

orbe

displaced by

an

unwinding activity. Unwinding

of

double-stranded DNA or RNA is an essential process in many cellular

functions, including replication,

recombina-tion, repair, transcriprecombina-tion,

mRNA

splicing,

and initiation of translation of mRNAs. This task is

generally accomplished

by NTP-dependent

DNAor RNAhelicases which

translo-cate

along

onestrand of

duplex

DNAorRNA

coupled

tothe

hydrolysis

of

ATP,

resulting

in

unwinding

of the

duplex.

Inthis

study

we

investigated

the RNA

duplex unwinding

ability

of

poliovirus

RNA-dependent

RNA

polymerase,

3DP0,

during

a chain

elongation

reaction in vitro. We showed that

poliovirus

polymerase

can

elongate through

a

highly

stable RNA

duplex

of over

1,000 bp.

This was

achieved

by unwinding

of the

duplex

and

displacing

the antisenseRNAfrom the

template.

The

unwinding activity

is an inherent property of the enzyme. It did not occurin the absence ofconcomitant RNA chain

elongation,

andit didnot

require

hydrolysis

of the

-y-phosphate

of ATP.Therefore,the

activity

exhibited

by poliovirus polymerase

is different from

thoseoftrueDNAorRNAhelicases whichmustharvest the energy from

hydrolysis

ofnucleotides in orderto drive the reaction

(10).

Since RNAduplex unwinding activityof

3DP"l

is

dependent

on

elongation,

it is

implied

that thedirection of

unwinding

occursfrom 3' to5'with respect tothetemplate

strand. Onthe basis ofourobservation thatonlyverysmall amounts of reaction intermediates accumulated during the course of the antisense

displacement

assay (Fig. 7A), the rateof

elongation

throughtheduplexregion does not appear

to differ much from that occurring on the single-stranded

region

of the

template.

This conclusion is supported by the fact thatantisense RNAswere

displaced

within 15 min of the reaction

(Fig.

7A,

lane

2),

which is consistent with the

elongation

rate reported in previous studies (31). Although

RNA

duplex

unwinding by

3DP01

occurs

quite efficiently,

the

role, if any, of this activity in RNA replication in vivo has notbeen demonstrated.

It was previously reported that partially purified EMCV RNApolymerase preparations from virus-infected cell ex-tracts were able to displace complementary RNA from a template in a reaction that was both elongation and ATP dependent (8). This unwinding activity was not consideredto be an inherent property of the EMCV polymerase since the activity was lost upon further purification of the enzyme. No restoration of the unwinding activity, however, was achieved byreconstituting different fractions from the puri-fication procedure. Therefore, one possible explanation for the apparent discrepancy with the findings reported in this study could be that the unwinding, but not the elongation property of the EMCV polymerase was inactivated during purification of the enzyme. Alternatively, the properties of the poliovirus and EMCV polymerases may be inherently different. For example, EMCV polymerase may be less processivethan thepoliovirusenzymeand therefore may be unable toelongate through a duplex region of thetemplate. To circumvent this problem, EMCV may have evolved to utilize an RNA helicase(either viral or cellular) as part of its replication machinery.

Most positive-stranded RNA viruses of both plants and animals encodeaprotein which contains nucleotide binding motifs and which appears to be involved in viral RNA replication(12-14).Theseproteinsappeartobehomologous tothe 2Cproteinsof thepicornaviruses,andthey have been postulated to function as NTP-dependent RNA helicases. Thecylindrical inclusion (CI) protein of plum pox potyvirus (apositive-strand RNA plant virus), which contains the NTP binding motif, has been demonstrated to have a nucleic acid-stimulated ATPase activity and an ATP-dependent RNAhelicaseactivity (19, 20). AlthoughCIproteinhas been

postulated to be involved in RNA replication, the exact biochemical function(s) of this protein has not been eluci-dated. From site-directed mutagenesis studies (27, 45), the nucleotide binding motifs of poliovirus 2C have been

sug-gestedtobeimportantin viral RNAreplication.At present, however, no biochemical evidence that 2C is an

ATP-dependentRNAhelicase has been demonstrated.

Hayes and Buck (15) have reported previously that puri-fied RNA-dependentRNApolymerase of cucumber mosaic viruscatalyzesthecomplete replication of cucumber mosaic virus genomic RNA (i.e., synthesis of progeny plus strand froma plus-strand template provided in the reaction). Cu-cumber mosaic virus polymerase contains two virus-en-codedpolypeptides(laand2a)and onehostpolypeptide. On the basis of the observation that the la subunit of the

polymerase contains sequence motifs characteristic of nu-cleic acid helicases, the investigators suggested that the

complete replicationof viral RNA waspossible because of a helicase activity present in the purified polymerase. They also suggested that 2C has an analogous function in the processofpoliovirus RNA replication. To date, no in vitro systemhas beendeveloped which can catalyze the complete replication of poliovirus RNA. Since we have shown that duplex RNA does not

impose

any noticeable resistance to chain elongation by

3DP",

we suggest that the inability to reconstitute an in vitro system which can catalyze the complete replication reaction is likely because of problems in initiation rather than elongation.

The observation that the poliovirus polymerase by itself can unwind an RNA duplex during the elongation reaction doesnotexclude the possibility that 2C is an RNA helicase. The molecular mechanism ofinitiation of RNA synthesis is

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still unknown; for example, it has not been determined what components are involved, what the order of reactions is, or how the synthesis of negative strands differs from that of the positive strands. Since 3DPo1 is the only protein known to be required and sufficient for the elongation reaction in vitro, it is possible that 2C is involved in RNA unwinding during the initiation of RNA synthesis and 3DPo1 unwinds RNA duplex during chain elongation.

The RNA duplex unwinding activity exhibited by poliovi-rus polymerase during chain elongation does not appear to be a common feature shared by all polymerases, since AMV RT did not unwind the same substrate used for poliovirus polymerase. Different polymerase systems appear to have evolved alternate mechanisms to solve the problem of du-plex formation between the template and the nascent strand during nucleic acid synthesis. Several reports have shown that the bacteriophage +6, which has three segments of double-stranded RNA as its genome, transcribes semicon-servatively such that one of the parental strands of each duplex is displaced by a newly made strand (46, 48). This scheme appears to be quite similar to that of the elongation reaction by polioviruspolymerase described in this study in which nascent strands form duplex structures with the template while displacing the prehybridized antisense RNA. It is not known whether the 46 polymerase is sufficient for this strand displacement or requires another auxiliary pro-tein(s). In the case of the transcription reactioncatalyzed by eukaryotic RNA polymerase II, a nascent RNA strand is prevented from hybridizing to the template DNA by the polymerase because of the enzyme's ability to bind the nascenttranscript (35). According to the model, the nascent RNA enters a product binding site on the protein surface while the transcription bubble advances along with the polymerase which thereby prevents formation of product-template duplex. In the case of bacteriophage Q3, the template RNA rather than the polymerase is responsible for the prevention of duplex RNA. According to the proposed mechanism, both the template strand and the nascent RNA product are displaced fromintermolecular base pair interac-tions in favor of the formation of local intramolecular base pairing (26, 34). This hypothesis has been supported by the observation that RNAs that form less stable intramolecular structures have a greater tendency to form extended RNA duplexes duringreplication, resulting in reduced synthesis of new RNA strands (1).

As expected from previous data (6), RT did not elongate through the region of the template which was hybridized to acomplementary RNA strand. During a retrovirus replica-tion cycle, a complementary DNA strand is synthesized from the viral genomic RNA, generating an RNA-DNA duplex. Subsequently, the RNA strand of the duplex is digested by the RNase Hactivity of RT, andgenomic DNA is synthesized. RT can unwind and displace RNase H hydrolysis products from a DNA template (6), although it was unable to unwind RNA-RNA duplex. Thus, separation ofproduct fromtemplate occursby thecombined action of RNase H and an unwinding-likeactivity of RT.

This study demonstrates that poliovirusRNApolymerase possesses an unwinding activity whichdisplacesextensively

base-paired segments of RNA from its template preceding

progression of a replication fork, allowing basepairing of the nascent strand to the template. The role of this activity

during replication of poliovirus RNA is unknown; it may removesecondary structure in thetemplateorit mayunwind duplex segments of different strands of RNA. Clearly, the release of template from product RNA strands to allow

furtherrounds ofsynthesisappears to be a requirement for thereplicationreaction.

ACKNOWLEDGMENTS

This work was supported by grant AI 17386 from the National Institutes of Health. T.M.D. was a recipient of a short-term Fogarty InternationalFellowship.

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Figure

FIG. 1.waspolioviruspGEM-4transcripts.digestedrusindicated Construction of the plasmids used for synthesizing RNA A BamHI-SalI fragment of pPOV-3 containing the sequence was ligated into the multiple cloning site of to generate pPV2C*3D
FIG. 2.Thepolaritytively.sequentrusdenotesequences,subsequent Diagram of transcripts used to generate RNA substrates
FIG. 3.visualizedbyThesesubstratesusedstrandedis indicated Substrates for RNA duplex unwinding assay
FIG. 5.markers.estimatedproductsphoresistemplatestratesindried a RNA synthesis by poliovirus polymerase
+2

References

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